Numerous reagents have been employed in methods for the determination of urea or urea nitrogen in biological fluids such as serum, plasma, urine, or the like. A widely accepted specific method involves incubation of the fluid with urease to bring about the enzymatic release of ammonia, which is then determined quantitatively by nesslerization. The urease nesslerization technique is time consuming and there is a need for more rapid methods. Several rapid methods are in use in which the amount of urea or urea nitrogen in a sample of biological fluid is determined by contacting the biological fluid with a reagent which combines with urea in the biological fluid to form a colored reaction product or chromophor; the depth or intensity of the color produced being proportional to the amount of urea present in the sample of biological fluid. The concentration of urea nitrogen in the sample is then determined by measuring the depth or intensity of the color, usually with a colorimeter or a spectro-photometer. By use of conversion charts or comparisons to standard solutions, the measurement of the color produced by the use of the reagent can be converted to give the concentration of urea nitrogen in the sample.
The diacetylmonoxime reaction has been used for the determination of urea since 1939. See Fearon, Biochem J. 33, 902 (1939). When urea is heated with diacetylmonoxime in the presence of a strong acid such as sulfuric, phosphoric, hydrochloric, or nitric a chromophor is formed having an absorption peak at 480 nm. See Natelson, et al., Am. J. Clin. Path. 21, 275 (1951); LeMar, et al., Analytical Chem. 29, 1233 (1957); Veniamin, et al., Clin. Chem. 16, 3 (1970); Lugosi, et al., Clin. Biochem. 5, 171 (1972); and Kitamura, Clin. Chem. Acta 4, 701 (1959). However, the color developed did not give a linear relationship between urea nitrogen concentration and absorbances. Coulombe and Faureau, Clin. Chem. 9, 102 (1963), introduced the use of thiosemicarbazide in conjunction with diacetylmonoxime for serum urea nitrogen determination. The sensitivity of the method was increased, but a different chromophor was produced which had an absorption peak at approximately 535 nm. This reaction required a long heating time (20 minutes or longer), a strong acid media (20% or higher), and the use of a protein-free serum filtrate. At approximately the same time the use of antipyrine was introduced in conjunction with diacetylmonoxime in a strong acid media for the determination of serum urea nitrogen. Ceriotti, et al., Clin. Chem. Acta 8, 295 (1963). This increased method sensitivity and produced a linear relationship between the urea nitrogen concentration and the absorbance readings. However, the procedure and its subsequent modifications, produced a third chromophor having a color peak at 460 nm.
The reaction between urea and reagents containing diacetylmonoxime and thiosemicarbazide has had numerous modifications. Marsh, et al., Clin. Chem. 11, 624 (1965), incorporated ferric ion into the reagent to intensify the color produced at about 535 nm and at the same time to allow a decrease in the acid concentration. The effect of metallic ions on the urea-diacetylmonoxime-phenazone reaction was described in Ceriotti, et al., Clin. Chem Acta 11, 519 (1965). Wybenga et al., Clin. Chem. 17, 891 (1971), stabilized the color by adding cadmium sulfate to the reaction mixture. However, all the diacetylmonoxime-thiosemicarbazide methods for urea nitrogen determination show deviation of linearity when the optical density of the color developed approaches a value of about 0.4-0.5 absorbance units.
An additional reference of interest is U.S. Pat. No. 3,567,374.